Kaposi's sarcoma-associated herpesvirus (KSHV) is the responsible agent for kaposi's sarcoma (KS), primary effusion lymphoma and multicentric Castlemans disease. A recent discovery revealed KSHV expresses multiple microRNAs with the potential to modulate host gene expression. Most microRNAs repress target gene expression by destabilizing the mRNA transcript and decreasing translational efficiency. The general path of research in our group starts with target prediction using expression profiling data (searching for microRNA-induced inhibition of gene expression), followed by target validation using luciferase reporters and protein expression measurements, and finally we conduct target significance assays. The significance assays attempt to address the question of why the virus has selected specific human target genes for inhibition by viral microRNAs. We hope to discover new functions of human genes as they relate to viral infection and cancer. Using previously generated expression profiling data, we constructed a new dataset utilizing a different method of expression analysis to integrate the expression data from multiple gain and loss of microRNA function experiments. After making new unbiased systematic microRNA target predictions based on the new method of expression analysis and disregarding whether these targets are predicted by commonly used microRNA target prediction programs, we cloned around fifty 3'untranslated regions (these are common microRNA target regions) from the predicted target genes into luciferase reporter plasmids. We have tested forty-nine predicted targets genes and twenty eight microRNA target genes were significantly inhibited using luciferase assays this year. We currently have a 57% success rate in accurate microRNA target prediction. This success rate is higher than common bioinformatic tools. In addition, we identified multiple examples of individual target genes being inhibited by multiple KSHV microRNAs. It is noteworthy to state approximately half of these microRNA:target interactions are not detected using bioinformatic methods which only search for sequence complementarity between the microRNA and potential mRNA target. A subset of these target genes has been further validated by looking at protein expression of endogenous target genes in response to viral microRNA expression, microRNA inhibition in infected cells and KSHV infection. To assess changes in protein expression, we utilize a near-infrared scanner to perform simultaneous two-color quantitative western blotting assays. In addition, we have mapped at least functional microRNA target sites in at least four different human genes using site directed mutagenesis. Furthermore, using KS biopsies we have determined multiple microRNA target genes that are inhibited in our cell culture systems are also inhibited at sites of KSHV infection in patients. In order to complement our mRNA expression profiling dataset, we have almost completed a proteomic screen to assess changes in protein expression as a result of KSHV microRNA expression. We have integrated the proteomic data with our microarray dataset. Our initial analysis identified a published KSHV microRNA human target gene in addition to other target genes. We are utilizing changes in protein expression as an additional filter in our microRNA target prediction method. We have used multiple methods to analyze the functional significance of the validated targets genes. Many targets are involved with regulation of the cell cycle, apoptosis and other cancer-related functions. Our progress studying the functional significance of KSHV microRNAs have revealed a cytokine receptor targeted by a KSHV microRNA with interesting functional consequences. We demonstrate this KSHV microRNA can inhibit cytokine-induced apoptosis and also suppress a pro-inflammatory cytokine response. This represents a mechanism for KSHV to avoid death of an infected cell during latency and to inhibit immune responses by the host. This data is under review for publication. Finally, we are actively studying the functional consequences of at least four other human genes that are targeted by KSHV microRNAs.